Protein kinase C comprises a family of more than 10 isozymes involved in the regulation of cell signalling. They have been grouped into 3 subclasses according to their structure and regulation. Classical or Ca2+ (cPKC), novel or Ca2+-independent (nPKC) and atypical or diacylglycerol (DAG)/phorbol ester-independent (aPKC). The first two types respond to the release of the second messenger DAG, which recruits the enzymes from their inactive cytosolic location to the membrane where they become allosterically activated. A similar effect can be achieved pharmacologically with the phorbol esters, which have substantially higher binding affinity for PKC than the DAGs. It is for that reason, that the phorbol esters have become the preferred agents to study the effects of PKC activation. On the other hand, we have synthesized a set of rationally designed DAG-lactones, which despite their structural simplicity, behave as highly potent PKC ligands capable of displaying comparable, albeit distinct activities to that of the phorbol esters, sometimes even surpassing them in potency. From a library set of more than 300 compounds we have selected a candidate DAG-lactone that showed potent but non-discriminating activity between isozymes alpha (cPKC) and delta (nPKC) in terms of in vitro binding and activation. However, under more physiological conditions in whole cells, we have been able to demonstrate that this compound shows unique isozyme specificity, which is determined by the selective targeting of specific isozymes to different intracellular compartments. Previous work has shown that there is an overlapping role for PKC-alpha and PKC-delta in the apoptotic effect on PMA in LNCaP cells. LNCaP cells are unique in that they only express the classical PKC-alpha, the novel PKC-delta and the atypical PKCs zeta and lambda. Here we show that PMA and the DAG-lactone activate a different subset of PKC isozymes to promote apoptosis in LNCaP cells. Specifically, the new DAG-lactone induced exclusive tranlocation of PKC-alpha to the cellular membrane and PKC-delta to the nuclear membrane. Although multiple PKC isozymes may contribute to the apoptotic effect, the DAG-lactone appears to operates exclusively through the alpha isozyme. Indeed, while the specific cPKC inhibitor G 6976 completely blocked the apoptotic effect of the DAG-lactone, and only partially that induced by PMA, the specific PKC-delta inhibitor rottlerin was ineffective in countering the apoptosis induced by the DAG-lactones while it was able to limit PMA-induced apoptosis. This novel DAG-lactone is the first known example of a selective activator of a classical PK-C isozyme in a cellular model. Further studies with other DAG-lactones promise to be useful in designing new tools to dissect isozyme-specific functions in cells. Finally, given the growth inhibitory properties of some PKC isozymes and their involvement in apoptosis, an emerging theme is that PKC activation rather than PKC inhibition may be of therapeutic value. Protein kinase C isozymes. Chimaerins. Activation/inhibition, Drug design. Zinc finger. Apoptosis, cellular localization.